RRC ID 3314
Author Iwaki H, Muraki T, Ishihara S, Hasegawa Y, Rankin KN, Sulea T, Boyd J, Lau PC.
Title Characterization of a pseudomonad 2-nitrobenzoate nitroreductase and its catabolic pathway-associated 2-hydroxylaminobenzoate mutase and a chemoreceptor involved in 2-nitrobenzoate chemotaxis.
Journal J Bacteriol
Abstract Pseudomonas fluorescens strain KU-7 is a prototype microorganism that metabolizes 2-nitrobenzoate (2-NBA) via the formation of 3-hydroxyanthranilate (3-HAA), a known antioxidant and reductant. The initial two steps leading to the sequential formation of 2-hydroxy/aminobenzoate and 3-HAA are catalyzed by a NADPH-dependent 2-NBA nitroreductase (NbaA) and 2-hydroxylaminobenzoate mutase (NbaB), respectively. The 216-amino-acid protein NbaA is 78% identical to a plasmid-encoded hypothetical conserved protein of Polaromonas strain JS666; structurally, it belongs to the homodimeric NADH:flavin mononucleotide (FMN) oxidoreductase-like fold family. Structural modeling of complexes with the flavin, coenzyme, and substrate suggested specific residues contributing to the NbaA catalytic activity, assuming a ping-pong reaction mechanism. Mutational analysis supports the roles of Asn40, Asp76, and Glu113, which are predicted to form the binding site for a divalent metal ion implicated in FMN binding, and a role in NADPH binding for the 10-residue insertion in the beta5-alpha2 loop. The 181-amino-acid sequence of NbaB is 35% identical to the 4-hydroxylaminobenzoate lyases (PnbBs) of various 4-nitrobenzoate-assimilating bacteria, e.g., Pseudomonas putida strain TW3. Coexpression of nbaB with nbaA in Escherichia coli produced a small amount of 3-HAA from 2-NBA, supporting the functionality of the nbaB gene. We also showed by gene knockout and chemotaxis assays that nbaY, a chemoreceptor NahY homolog located downstream of the nbaA gene, is responsible for strain KU-7 being attracted to 2-NBA. NbaY is the first chemoreceptor in nitroaromatic metabolism to be identified, and this study completes the gene elucidation of 2-NBA metabolism that is localized within a 24-kb chromosomal locus of strain KU-7.
Volume 189(9)
Pages 3502-14
Published 2007-5-1
DOI 10.1128/JB.01098-06
PII JB.01098-06
PMID 17277060
PMC PMC1855914
MeSH 3-Hydroxyanthranilic Acid / metabolism Amino Acid Sequence Amino Acid Substitution Aminobenzoates / metabolism* Binding Sites / genetics Chemotaxis Cloning, Molecular Escherichia coli / genetics Escherichia coli / metabolism Gene Deletion Gene Expression Intramolecular Transferases / chemistry Intramolecular Transferases / genetics* Intramolecular Transferases / metabolism* Models, Biological Models, Molecular Molecular Sequence Data Mutation Nitrobenzoates / metabolism* Nitroreductases / chemistry Nitroreductases / genetics* Nitroreductases / metabolism* Protein Structure, Tertiary Pseudomonas fluorescens / enzymology Pseudomonas fluorescens / genetics Pseudomonas fluorescens / metabolism* Pseudomonas fluorescens / physiology Pseudomonas putida / genetics Sequence Analysis, DNA Sequence Homology, Amino Acid
IF 3.234
Times Cited 41
WOS Category MICROBIOLOGY
Resource
Prokaryotes E. coli pK18mobsacB ME9042(BL21) CV-25(DH5alpha) CV-61(S17-1)
INFORMATION E.coli